Histone deacetylase inhibitor belinostat represses survivin expression through reactivation of transforming growth factor beta (TGFbeta) receptor II leading to cancer cell death.

Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor ß receptor II (TGFßRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFß signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFß signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFß/protein kinase A signaling pathway. Induction of TGFßRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFßRII might potentially benefit from the use of this histone deacetylase inhibitor.

Selective Inhibition of Histone Deacetylases 1/2/6 in Combination with Gemcitabine: A Promising Combination for Pancreatic Cancer Therapy.

Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of <10% due in part to a lack of effective therapies. Pan-histone deacetylase (HDAC) inhibitors have shown preclinical efficacy against PDAC but have failed in the clinic due to toxicity. Selective HDAC inhibitors may reduce toxicity while retaining therapeutic efficacy. However, their use requires identification of the specific HDACs that mediate the therapeutic effects of HDAC inhibitors in PDAC. We determined that the HDAC1/2/3 inhibitor Mocetinostat synergizes with the HDAC4/5/6 inhibitor LMK-235 in a panel of PDAC cell lines. Furthermore, while neither drug alone synergizes with gemcitabine, the combination of Mocetinostat, LMK-235, and gemcitabine showed strong synergy. Using small interfering (si)RNA-mediated knockdown, this synergy was attributed to inhibition of HDACs 1, 2, and 6. Pharmacological inhibition of HDACs 1 and 2 with Romidepsin and HDAC6 with ACY-1215 also potently synergized with gemcitabine in a panel of PDAC cell lines, and this drug combination potentiated the antitumor effects of gemcitabine against PDAC xenografts in vivo. Collectively, our data show that inhibition of multiple HDACs is required for therapeutic effects of HDAC inhibitors and support the development of novel strategies to inhibit HDACs 1, 2, and 6 for PDAC therapy.

Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity.

The genomic structure and transcriptional regulation of the rat apelin receptor (APJR) were analysed by rapid amplification of 5' cDNA ends (5'-RACE), transient expression assays and DNA-protein interaction. Analysis of the 5'-flanking region of a rat genomic clone shows no TATA box, but a putative CAAT box and several putative binding sites for transcription factors are present. Two transcriptional start sites were identified by 5'-RACE, RNase protection and primer extension analyses. Promoter activity was exhibited in the APJR- expressing SH-SY5Y cell line as well as in COS-7 and Chinese hamster ovary (CHO-K1) cells. Consecutive 5'-deletion analysis revealed the highest promoter activity in a region between bp -966 and -165. DNaseI footprint analysis revealed seven protected regions and electrophoretic mobility shift, super-shift and competition assays identified individual DNA-protein complexes capable of binding Sp1, estrogen receptor (ER)alpha, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)gamma transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.

The role of transforming growth factor alpha in determining growth factor independence.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.

Activation of the TGFalpha autocrine loop is downstream of IGF-I receptor activation during mitogenesis in growth factor dependent human colon carcinoma cells.

The inappropriate expression of TGFalpha in growth arrest contributes to malignant progression in human colon carcinoma cells. Early stage, non-progressed colon tumor cells show a down-regulation of TGFalpha in growth arrest and require both nutrients and growth factors for re-entry into the cell cycle. In contrast, highly progressed cells up-regulate TGFalpha during growth arrest and require only nutrients for re-entry. Given the importance of TGFalpha in malignant progression, this work addressed the regulation of TGFalpha expression in the early stage colon carcinoma cell line, FET. Growth-arrested FET cells down-regulated the expression of TGFalpha, EGFr and, in turn, EGFr activation. These quiescent cells continued to express high levels of IGF-IR protein, but IGF-IR activation was undetectable. Cell cycle re-entry required exogenous growth factor activation of the IGF-IR by insulin or IGF-I. This IGF-IR activation resulted in S phase re-entry and was accompanied by an approximate threefold induction of TGFalpha expression along with EGFr activation at 1 h following release from growth arrest. Activation of IGF-IR occurred within 5 min of cell-cycle re-entry. Previously identified DNA binding proteins which bind to a unique TGFalpha/EGF response element within the TGFalpha promoter were similarly induced following IGF-IR activation. The addition of EGFr neutralizing antibodies abolished the activated IGF-IR stimulated S phase re-entry. Moreover, disruption of the growth arrest associated down-regulation of TGFalpha in FET cells by constitutive TGFalpha expression abrogated the requirement for IGF-IR activation for cell cycle re-entry. Consequently, this study indicates, for the first time, that IGF-IR activation up-regulates components of the TGFalpha autocrine loop resulting in TGFalpha-mediated EGFr activation which was critical for IGF-IR mediated re-entry into the cell cycle from the growth-arrested state.

Ron knockdown and Ron monoclonal antibody IMC-RON8 sensitize pancreatic cancer to histone deacetylase inhibitors (HDACi).

Recepteur d'origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer.

Identification of a novel TGFß/PKA signaling transduceome in mediating control of cell survival and metastasis in colon cancer.

BACKGROUND: Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFß tumor suppressor activities in the metastatic process is essentially unknown. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing in vitro and in vivo techniques, we have shown that loss of TGFß tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFß receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFß/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFß/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFß receptor restoration with reactivation of the TGFß/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival. CONCLUSION/SIGNIFICANCE: This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFß function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFß signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFß/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors.

Epigenetic Targeting of Transforming Growth Factor ß Receptor II and Implications for Cancer Therapy.

The transforming growth factor (TGF) ß signaling pathway is involved in many cellular processes including proliferation, differentiation, adhesion, motility and apoptosis. The loss of TGFß signaling occurs early in carcinogenesis and its loss contributes to tumor progression. The loss of TGFß responsiveness frequently occurs at the level of the TGFß type II receptor (TGFßRII) which has been identified as a tumor suppressor gene (TSG). In keeping with its TSG role, the loss of TGFßRII expression is frequently associated with high tumor grade and poor patient prognosis. Reintroduction of TGFßRII into tumor cell lines results in growth suppression. Mutational loss of TGFßRII has been characterized, particularly in a subset of colon cancers with DNA repair enzyme defects. However, the most frequent cause of TGFßRII silencing is through epigenetic mechanisms. Therefore, re-expression of TGFßRII by use of epigenetic therapies represents a potential therapeutic approach to utilizing the growth suppressive effects of the TGFß signaling pathway. However, the restoration of TGFß signaling in cancer treatment is challenging because in late stage disease, TGFß is a pro-metastatic factor. This effect is associated with increased expression of the TGFß ligand. In this Review, we discuss the mechanisms associated with TGFßRII silencing in cancer and the potential usefulness of histone deacetylase (HDAC) inhibitors in reversing this effect. The use of HDAC inhibitors may provide a unique opportunity to restore TGFßRII expression in tumors as their pleiotropic effects antagonize many of the cellular processes, which mediate the pro-metastatic effects associated with increased TGFß expression.

Vasopressin potentiates corticotropin-releasing hormone-induced insulin release from mouse pancreatic beta-cells.

Arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) have both been implicated in modulating insulin secretion from pancreatic beta-cells. In the present study, we investigated the insulin-secreting activities of AVP and CRH in wild-type and AVP VIb receptor knockout mice. Both neuropeptides stimulated insulin secretion from isolated mouse pancreatic islets. The response of islets to CRH was increased fourfold by concomitant incubation with a subthreshold dose of AVP that alone did not stimulate insulin secretion. Activation of the endogenously expressed M3 receptor by the cholinergic agonist carbachol also potentiated CRH-induced insulin secretion, indicating that the phenomenon may be pathway specific (i.e. Ca2+-phospholipase C) rather than agonist specific. The protein kinase C (PKC) inhibitors Ro-31-8425 and bisindolylmaleimide I attenuated the potentiating effect of AVP on CRH-stimulated insulin secretion and blocked AVP-stimulated insulin secretion. A possible interaction between the PKC and protein kinase A pathways was also investigated. The phorbol ester phorbol myristate acetate (PMA) stimulated insulin secretion, while the addition of both PMA and CRH enhanced insulin secretion over that measured with either PMA or CRH alone. Additionally, no AVP potentiation of CRH-stimulated insulin secretion was observed upon incubation in Ca2+-free Krebs-Ringer buffer. Taken together, the present study suggests a possible synergism between AVP and CRH to release insulin from pancreatic beta-cells that relies at least in part on activation of the PKC signaling pathway and is dependent on extracellular Ca2+. This is the first example of a possible interplay between the AVP and CRH systems outside of the hypothalamic-pituitary-adrenal axis.